There is a fundamental gap in our understanding of how HIV-1 maintains latency in astrocytes. This gap denotes a significant problem, because, unless this gap is filled, understanding the molecular mechanisms of HIV-1 latency leading to HIV-associated dementia (HAD) cannot be achieved. Furthermore, the knowledge required to develop novel therapeutic strategies for HAD patients will remain largely unknown. The long-term goal is to understand the molecular mechanisms that contribute to HIV-1 latency in the brain and develop a therapeutic strategy to combat HIV. The objective of this application is to determine the role of Natural Killer Lytic-Associated Molecule (NKLAM) in the post-transcriptional regulation of HIV-1 gene expression. The central hypothesis of this application is that NKLAM synergizes with Sam68 and alleviates a post-transcriptional block to HIV-1 production in astrocytes. The hypothesis has been formulated from the strong preliminary data, demonstrating that NKLAM interacts with Sam68, which in turn, regulates HIV-1 Rev function in astrocytes. The rationale for the proposed project is that, once the role of NKLAM in the HIV-1 life cycle is understood, its production can be modulated in such a way that impacts HIV-1 production in the prevention and treatment of AIDS, including HAD. Guided by the preliminary data, the central hypothesis will be tested to accomplish the objective of this application by pursuing the following two specific aims: 1) Determine the functional significance of NKLAM in HIV-1 replication. The working hypothesis is that NKLAM relieves a post-transcriptional block to HIV-1 production in astrocytes. Under this aim, stable NKLAM expressing astrocytes will be created and assessed for the alleviation of Rev block and HIV-1 production by examining viral mRNA export. 2) Investigate the mechanism of action of NKLAM in Sam68/RRE-mediated transactivation. The working hypothesis is that NKLAM must act in the nucleus to synergize with Sam68. Employing mutational, microscopic and ubiquitination analyses, the mechanism of action of NKLAM in Sam68/Rev/RRE function will be determined. The proposed work is innovative, because, so far, no E3 ubiquitin ligase(s), specifically NKLAM, has been implicated in the post-transcriptional regulation of HIV-1 gene expression. Most importantly, to date, there are no published reports implicating, any E3 ligase(s), particularly, NKLAM- mediated Sam68 ubiquitination in alleviating the Rev block in astrocytes. The proposed research is significant because it is expected to provide the knowledge needed to develop novel strategies to eliminate HIV-1 reservoirs and combat HAD. PUBLIC HEALTH RELEVANCE: The proposed research is relevant to public health, because, determining the role of NKLAM in HIV-1 replication is eventually expected to increase our understanding of the molecular basis of HIV-1 latency operating at the level of post-transcription in astrocytes. Therefore, the proposed research is relevant to the part of NIH's mission that relates to the knowledge needed to develop novel therapeutic strategies for HAD patients.